Insulin derivatives crosslinked by a dicarboxylic acid moiety

ABSTRACT

Insulin derivatives in which the A-1 and B-29 free amino groups are crosslinked by a bridge of the formula - CO- X- CO- in which X is a carbon-carbon bond or an aliphatic group are described together with their method of preparation and use.

United States Patent [191 Brandenburg et al.

[ 1 Sept. 23, 1975 INSULIN DERIVATIVES CROSSLINKED BY A DICARBOXYLIC ACID MOIETY [75] Inventors: Dietrich Brandenburg, Schmithof,

uber Aachen; Walter Puls, Wuppertal, both of Germany [73] Assignee: Bayer Aktiengesellschaft, Germany [22] Filed: Feb. 16, 1973 [21] Appl. No.: 333,361

[30] Foreign Application Priority Data Mar. 1, 1972 Germany 2209835 52 U.S.C1 ..260/l12.7;7424/178 51 Int. c1. A61K 37/26; c07c 103/52 58 Field ofSearch ..260/112.7

[56] References Cited UNITED STATES PATENTS 3,679,653 7/1972 Schuck et al. 260/1 12.7

FOREIGN PATENTS OR APPLICATIONS 1,584,535 12/1969 France OTHER PUBLICATIONS Kagedal et al.; Acta Chem. Scan, 25, 1855-1859 (I971).

Csorba et al.; Horm. Metab. Res., 2, 305 (1970); from Chem. Abstr. 74:38373q.

Primary ExaminerLewis Gotts Assistant Examiner Reginald J. Suyat [57] ABSTRACT 27 Claims, 4 Drawing Figures US Patent Sept. 23,1975 Sheet 1 of2 3,907,763

US Patent Sept. 23,1975 Sheet 2 of 2 3,907,763

FIG. 3

FIG. 4

INSULIN DERIVATIVES CROSSLINKED BY A DICARBOXYLIC ACID M'OIETY The present invention pertains to new nionomericinsulin derivatives in which the A and B chains are crosslinked via the A-1 and 8-29 amino groups. to their preparation, and to theirmedicinal use as antidiabetic agents.

It is known that insulin can be chemically modified by means of various monofunctional reagents; Some of these derivatives can be isolated in the homogeneous form and possess modified biological properties. see. c.g., Levy ct al.. Biochemistry 6. 3559 (l967 Lindsay et al., Biochem J. l2l. 737 (l97l and Africa et al.. Biochemistry 9, I962 1970). In the reaction of insulin with bifunctional reagents on the other hand. it has generally hitherto been possible to obtain only heterogcneous'mixtures without a single reaction product: 'see e.g.. DDR Pat. No. 10,002; Zahn et al.. Makromol. Chem. 26, 153 (1958). In these cases. the bifunctional reagent appears to react with the various functional groups of the insulin molecule. In addition to monomeric derivatives. higher-molecular products with an indeterminate degree of polymerization are also produced. Moreover the known homogeneous insulin derivatives with an intramolecular bridge. such as the m-phenylenedithiocarbamoyl bridge. have proved to be biologically inactive; see Brandenburg et al., Diabetes, Proc. 7th Congress Int. Diabetes Fed. Buenos Aires, 1970. Exeerpta Medica Int. Congr. Series 231. 363 i971 g The present invention provides new biologically active insulin derivatives in which the a-amino group of glycine of the A-chain is bifunctionally crosslinked to the e-amino group of lysine of the B-chain by a group of the formula:

Ll ll in which X is a direct carbon-carbon bond. or an aliphatic hydrocarbon chain with 1-15 C atoms, of which one or more carbon atoms are optionally replaced by one or more hetero atoms or groups of hetero atoms. and either unsubstituted or substituted by hydrophilic groups.

This invention also provides a process for the production of the compounds defined above in which insulin is reacted with an activated derivative ofa dicarboxylic acid of the general formula:

ll ll 0 o in which X is as defined above, and

Y is a radical that activates the carboxylic acid groups of the acid.

in a polar organic solvent or in a mixture of an organic solvent and water. in the presence of a proton acceptor.

The new compounds thus produced are preferably purified to remove accompanying by-products and other impurities by a separation process which differentiates the products firstly according to molecular weight and secondly according to their charge. Preferably this separation process is followed by a precipitation of the new compound in crystalline form from a solution containing a zinc salt.

Only intramolccularly crosslinked insulin derivatives in which the amino groups of glycine A-1 and lysine 8-29 are linked are formed in the crosslinking reaction according to the invention. It is thus possible to dispense with the temporary protection of the B-1 (phenylalanine) -amino groups. even though it could have been expected. from the state of the art. that this amino group would also be substituted to a high degree; see .e.g.. Lindsay et al.. Biochem. J. 121. 737 (i971); Levy ct al.. Biochemistry 6. 3559 (1967). It is equally unex- Le n YS a c oLy Ala it wherein X is carbon-carbon bond or analkylene chain containing 1 to 15 carbon atoms, from to 2 of said carbon atoms beingreplaced by a corresponding number of sulfur, oxygen or imino groups, said alk'yl'ene chain being unsubstituted or substituted by one or two amino groups.

The above depiction of the insulin molecule has been modified from the conventional format for graphic convenience. it being understood the insulin molecule in fact is identical with the known structure save for the presence of the crosslinking bridge.

ln Formulas l and ll, X is preferably a carbon-carbon bond or an alkylene group of from 1 to l5 carbon atoms, most preferably, the group (CH where n has a value of l to 15. Y is preferably an optionally substituted phenoxy radical.

v lf beef insulin and the bis-p-nitrophenyl ester of adipic are used as the'starting substances, the course of the reactio'n' in the process of the invention can be repdimethylformamide are completely satisfactory.

The proton acceptor includes all the basic compounds which are customarily employed in peptide synthesis as. I for example. triethylamine or N- methylmorpholine. The latter base is preferably employed when'working with optically active carboxylic acids in order to avoid racemization. When working in the presence of water. it is also possible to use alkali metal, hydroxides. bicarbonates or carbonates. such as sodium bicarbonate sodium carbonate and the like.

Generally temperatures of between 0 and 40, prefresented in the specification by the following? erably l8 to"C. are 'used. The reactions are usually (A-l) I V I (A-Zl) H NG1y. .Cys-C ]'s'. .Cys .Cys -Asn S I S l l S S l y t l l H N-Ph.......';..Qys.'..';.........Cys..'. .Lys--A1a g-N0 0C0(CH C00 l)- NO I .(A-l) I l (A-Zl) HN-Gly. .ttys- Cys. .Cys. Asn

Suitable activated derivatives of dicarboxylic acids are known or can be readily manufactured according to known processes; see e.g., Zahn et al., Chem; Ber. 96, 1747 (I963); Sehnell et al Kolloid-Z. 203, 27 (1965 Zahn et al., Forschungsber. des Landes. Nordrhein-Westfalen. No. 1897, Brandt, Ed., Westdeutseher Verlag, Cologne and Opladen (1967). The preferred derivatives in which Y is asubstituted phenoxy group can be prepared from the dicarboxylic acid, the appropriately substituted phenol, and dicyclohexylcarbodiimide.

Examples of these activated derivatives include adipic acid bis-p-nitrophenyl ester, pimelic acid bis-N- hydroxysuccinimide ester, suberic acid bis-2,4,5- trichlorophenyl ester, sebacic acid bispentachlorophenyl ester and the like. Amino acids which possess two carboxyl groups in an activated form but in which the amino groups are protected, such as N,N'-bis-tert.-butoxyearbonyl'cystine-bis-2.45-

va *fv a...

HN-Phe.

carried out under normal pressure. It is advantageous, but not essential, to exclude light and oxygen by working in the dark under a protective gas such as nitrogen to avoid side-reactions such as oxidative damage to the insulin.

In carrying out the process, I to 2 moles, preferably 1.2 to 1.3 moles, of the activated dicarboxylic acid derivative are generally employed per each mole of crystal insulin or amorphous insulin. Conveniently the solution of the activated derivative is slowly added, dropwise, usually over the course of l to 5 hours, to the solution of the insulin. The reaction mixture is then allowed to stand for several hours longer. with stirring if necessary.

lnso'l ation can be effected by adding a solvent which is miscible with the reaction solvent and which effects precipitation of the insulin derivatives; e.g., methanol or ether. One can also dialyze the reaction solution against water and isolate the insulin derivative in this way. The mixture can be acidified, for example with acetic acid, tostop the reaction before the dialysis. Dialysis against dilute ammonium bicarbonate solution. in which residual activated groups of the dicarboxylic acid are aminolyzed. has also proved advantageous.

After dialysis. the product can be lyophilized directly or the deisred insulin derivative can be precipitated by adjusting the pH to the iso-electric point of the deriva- Live. A

The crude insulin derivative generally is then fractionated. either while moist or after drying. to separate the product in accordance with its molecular weight. Preferably this is accomplished by gel chromatography utilizing conditions under which insulin and the derivatives do not aggregate. For example. Sephadex G-50 fine resin in percent strength acetic acid can be used for this purpose. The preparation obtained after dialysis and freeze-drying is generally then further fractionated so as to separate molecules according to their charge. For this, ion exchange chromatography or electrophoretic processes can be used. Preferably, an ion exchange chromatography is employed in an acid mesulfitolysis A B (SS) (Insulin) dium. for example on SE-Sephadex A at pH 3.0 in

acetic/7 M urea. using a sodium chloride gradient.

The product obtained after dialysis, freeze-drying and. where necessary, desalination by means of gel fil- A B (SS) (1 ll 0 o (crosslinked insulin) tration. for example of Sephadex G 25, or by isoelectric reprecipitation can. if necessary, be further purified by subsequent ion exchange chromatography in a weakly alkaline solution. for example on DEAE-Sephadex A 25 at pH 8.4.

N N -suberoylinsulin (beef);

N N"- -a7.elaoylinsulin N- -L N -sebaeoylinsulin (beef, pig);

N- N' -undecanedioylinsulin (beef);

N N -dodecanedioylinsulin (beef);

N N -tridecanedioylinsulin (beef) N.-l-l naa N ,N'-bis-tert.-butoxycarbonyl cystinylinsulin (beef);

N-- N -L-cystinylinsulin (beef);

N N-benzyloxycarbonyl-L-glutamylinsulin (beef); and

N N -L-glutamylinsulin (beef)- The structure of the crosslinkcd insulin derivatives according to the invention can be demonstrated in a number of ways.

Insulin and all derivatives in which amino groups are monofunctionally substituted yield two products upon splitting the disulfide bonds, as for example by oxidative sulfitolysis Bailey et al., J. Biol. Chemistry 234. 1733, (1959). These two products thus correspond to separate A- and B-chains. in the form of S-sulfonates:

A(SS03 sso (A-chain). (B-chain) In the ease of an insulin derivative linked by means of a bifunctional bridge between glycine and phenylalanine or lysine only a single chain derivative is formed:

A (sso sulfitolysis O F i 0 C -B(SSO (crosslinked chain derivative) FIG. 1 shows four electropherograms for various cross-linked and degraded peptides.

FIG. 2 is theelution diagrams of a gel chromatography of azelaoylinsulin.

FIG. 3 is the elution diagram of an ion exchange chromatography of suberoyl-insulin.

FIG. 4 comprises the UV spectra of insulin and glutarylinsulin. I

As shown in FIG. 1, the compounds of Examples 1 to 4 and 6 to 9. upon being subjected to a sulfitolysis, showed only one band upon electrophoresis. The derivatives of'Examples I1 and 12 which are crosslinked with cystine gave two bands. as expected. since the cystine bridge is also split.

It can also be shown by enzymatic degradation and end group determination that the bridge is l'oeated'exclusively between glycine-' and lysine and not-between glycine and phenylalanine "Adipoyl A- chain-tetra-S-sulfonate-B-chain-dis-S-sulfonate is thus split by means of trypsin according to'Wang et Biochemistry 6. 2 l 196 7 Afterfreeze-drying. the products of-thc reaction are separated by paper-electrophoresis at pH 2 (2.4 M formic acid/7 M urea). -as shown in FIG. 1., No. 3.0111) the tvt'o'split pep'tid'es indicated in the following table are found. All other crosslinked insulins (Examples 1 to -4 and 6 m9) give the same results on electrophoretie analysis and end groupdetermination. The table which follows shows the' aminoacid compositionof'the derivative n. n"? ea'dipoyl- I A-chain-tetra-S-sulfonate-B-chain-bis-S-sulfonate*" (A-AD-'B') obtained frotn adipoylins'ulin byrbxidative sulfitolysis. and of the peptides obtained therefrom bysplit'ting with trypsin. N N adipOIyI-A-liainte't'ra-sulfonate-B( 23-30). which is' designated AA- DB(23-30). and B(l22)-bis-S-sulfonate which is designated B( 1-22). The aminoacid analysis was conducted according to the method of Moore and Stein. with a hydrolysis time of 48 hours at 1 10C. All values are uncorrected and relative to glycine.

+denotes that the substance is present but was not determined quantitatively. 13(23-30) aminoaeid sequence 23-30 of the ll-ehain Bl 1-22) amino-acid sequence 1-22 of the B-chain Referring now to the drawings. FIG. 1 shows four paper clectropherograms at pH 2 HCOQH/7 M urea) of:

l. A-chain (left and B-chain (right) in the S-sulfonate form;

2. N N -adipoyl-A-chain-tetra-S-sulfonate-B- chain-bis-S-sulfonate;

3. the split peptides obtained therefromby degradationby trypsin; and fl 4. the split peptides obtained from insulin B-chain-S- sulfonates with trypsin. 8 1-22 (left) and B (23-29) (right). a l v Colorationwas produced by means o f diazgotized sulfanilic acid. The arrow marks the starting ot the electrophoroses; I g :v v

F1032 shows the elution of chromatogra- (buffer: 2.4. M

phy of approximately 600 mg of the crudeazelaoylinsulin obtained in Example 8 on a 5 X cm column with Sephadex G-50 fine in 10% strengthacetic acid'using allow speed l00ml/hour and taking fractions of 12 ml. The abscisa corresponds to fraction number while the ordinate is extinction at.254 nanometers (nm); Fraction l contains oligomeric insulin derivatives., fraction 2 contains mainly dim-eric insulin derivatives and fraction.3.contains monomeric insulin derivatives. The .elution diagrams of the gelchromatography of the other bifunctit'mally"cross-linked insulin derivatives show a similar pattern.

FIG. 3 shows theclution diagram of the ion exchange chrotnatography of- 320 mg of-the crude suberoylinsulin obtained in Example 7. fraction 3. ona 1.5 X 45 cm column-with SE-Sephadex at-pH 3.0 with a linear sodium .chloride gradient and a .flow speed 30-35 -ml/hour., Fractions of 6.8 ml were'taken with extinction measured at 254 nm The abscissa corresponds to fraction number while the ordinate is theconcentration of sodium=chloride in moles/liter. Thefraction with maxi- -mum at fraction number 60 corresponds to insulin der-ivative.with the. suberoylbridge between glycine and lysine The .elution diagrams of the ion exchangev chromatography ofthe, other bifunctionally crosslinked insulin derivatives are similar.

FIG. 4 comprises the UV spectra of amorphous insulin at concentration 0.767 mg/ml (shown with a dotted line). and glutarylinsulin at concentration 0.636 mg/ml (shown with a solid line) in 0.05 M ammonium bicarbonate solution at pH 8.2. The ordinate is extinction and the abscissa is wavelength in nanometers The new compounds display a blood glucoselowering action and are therefore suitable for the treatment of diabetics in general and especially those who, as a result of the formation of antibodies against beef insulin and/or pig insulin-require high doses of conventional insulin preparations. but whose requirements are satisfactorily met, according-to experience. by lower doses after changing. over to a new preparation against which no antibodies are formed.

The present invention includes pharmaceutical compositions and medicaments in dosage unit form containing the new compounds. In common with known insulins and insulin derivatives. the new compounds are generally: administered by injection. Accordingly this invention provides a sterile aqueous injectable pharmaceutical composition containing a compound according to the invention. In a preferred form of this composition. the compound is in crystalline form in suspension in a solution of a zinc salt. Conventional additives may also be present.

This invention further provides a medicament in dosage unit form. The expression medicament in dosage unit form" as used in this specification means a discrete coherent article containing a predetermined individual quantity of the insulin derivative such that one article is utilized in a single therapeutic administration. A preferred example of a medicament in dosage unit form according to the invention is'an ampoule containing be conveniently observed in known laboratory models.

Thus insulin lowers the bloodglucose levels of rats and within limits. this action is (1 dependent. The insulin I I Insulin Active Compound Activity N N -oxalylinsulin (beef) 52 N N "-glutarylinsulin (beef) 32 N N ""-'"-adipoylinsulin (beef) 4l N N '"-pimeloylinsulin (beef) 35 N beroylinsulin (bccll I N oylinsulin (beef) I00 N t sch-acoylinsulin (beef) (\I N N ""-"-undecanedioylinsulin (beef) (v9 N N ""-"'-dodccancdioylinsulin (beef) 46 N 40 N '"-tridecancdioylinsulin (bee-I The following examples will serve to, further illustrate the production, by the process of the invention. of the present cross-linked insulin derivatives. These examples should not be construed as a limitation on the scope of the invention which is defined only by the appended claims. I

EXAMPLE I N N -0x'alylinsulin (beef) A solution of 39.8 mg I20 urnol) of oxalic acid bisp-nitrophenyl ester-in ml of dimethylsulfoxide was added dropwise over-the course of 4 hours at room temperature. with stirring, to a solution of 640 mg I00 82 mol) of crystalline beef insulin and ISO ul of triethylamine in 75 ml of dimethylsulf'oxi'de. The reaction solution was left to stand for a further 60 hours at room temperature and was then dialyzed first for 2 hours against running water and subsequently twice for one hour at a time against one liter of 0.05 M ammonium bicarbonate solution. The solution was adjusted to pH 4.9 with dilute hydrochloric acid and the protein which precipitated was collected by centrifugation and washed'with water. The moist productwas dissolved in 3 ml of glacial acetic acid and 1-7 ml of water and chromatographed on a column (5 X I50 em) with Sephadex G-50 fm e in strength acetic acid. The eluate was dialyzed in 3 fractions (compare FIG. 2), '4 times against I liter of distilled water at a-time, and subsequently lyophilized.

310 mg of fraction 3 we e dissolved in 3 ml of 1.5 M acetic acid/7 M urea/0.05 M sodium chloride (pH 3.0) and applied to a column (1.5 45 cm) with SE- Sephadex which wasequilibrated with the same buffer.

Elution was carried out by means of a lineargradient of 250 ml of starting buffer and'250 inl of added buffer which had the same composition as the starting buffer but contained 0.2 M sodium chloride. The eluate under the maximum (compare FIG. 3) was dialyzed 3 times for I hour at a time against 1 liter of distilled water and lyophilized. The residue was freed of the residual salt and urea by chromatography on a 3 X 60 cm column with Sephadex G-25 in 0.05 M ammonium bicarbonate solution and the eluate was lyophilized.

Yield: 98.2 mg (I571 of theory). 5278 5.490 (in 0.05 M ammonium bicarbonate solution. pH 8.2) (compare FIG. 5).

Paper electrophoresis at pH 2: single substance. R (electrophoretic mobility. relative to insulin) 0.74. Free amino groups using the dansyl chloride method of Gray. Methods in Enzymology. Volume I I I39. I I967) showed only phenylalanine.

Oxalylinsulin crystallizes in the form of small prisms from citrate buffer containing zinc ions. see Schlichtkrull. Aeta Chem. Scand. I0. 1455 I956).

EXAMPLE 2 N N -succinylinsulin (beef) 640 "mg of crystalline beef insulin were reacted over the course of 3 hours with 43.2 mg I20 umol) of succinic acid bis-p-nitrophenyl ester under the conditions described in Example I. The product was worked up and gel chromatography was carried out as therein described.

'Fraction I mg Fraction 2 I05 mg Fraction 3 4I I mg ((14.2% of theory) 410 mg of fraction 3 were ehromatographed on SE- Sephadex. and worked up as described in Example I. The main fraction (compare FIG. 3) after chromatography on Sephadex G-25 yielded I39 mg. (2I.7 /1 of Weighings:

Fraction I 54 mg Fraction 2 I mg Fraction 3' 330 mg (51.6% of theory) 320 mg of fraction 3 were chromatographed on SE- Sephadex. and worked up as described in Example I. after desalination by gel filtration on Sephadex G-25. followed by iso-electric reprecipitation at. pH 4.8. 110.3 mg( 17.3% of theory) of glutarylinsulin were obtained fromthe main fraction (compare FIG. 3).

e278 5,400 (in 0.05 M ammonium bicarbonate solution) Paper electrophoresis'at pH.-2: single substance R 0.74 I f Free amino groups: phenylalanine Crystal form: felted small needles.

. EXA-MPLE4 N adipoylinsulin (beef) A solution of 93.2 mg (240 uniol) of adipic acid bisp-nitrophenyl ester in5 mlof dimethylsulfoxide was added dropwise at room temperature with stirring to 1.27 g (200 ,umol) of crystalline beef insulin in 140 ml of dimethylsulfoxide in the presence of 0.3 ml of triethylamine. After standing for 60 hours at room temperature. the reaction solution was worked up as described in Example 1. Upon gel chromatography on Sephadex G-50 fine in strength acetic acidfthe following were obtained: r

Fraction 1 304 mg Fraction 2 335 mg Fraction 3 587 mg (46.2% ol'thcory) 580 mg of fraction 3 were ehromatographed in two equal parts on SE-Sephadex in the. manner described in Example 1. The main fractions-(compare FIG. 3) were combined after freeze-drying and desalinatedby gel filtration as described in Example l.

Yield: 294 mg (23% of theory) of adipoylinsulin 6276 5.650 (in 0.05 M ammonium bicarbonate so lution, pH 8.2)

Paper electrophoresis at pH 2: singe substance Free amino groups: phenylalanine Crystal form: small needles and spherical particles.

7.8 mg p.-mol) of adipic acid bis-p-nitrophenyl ester in 2.5 ml of dimethylsulfoxide were added dropwise over the course of 2 hours. with stirring to a solution of 120 mg (18.7 umol) of crystalline beef insulin in 15 ml of climethylsulfoxideand 11.1 of triethylamine. A further 7.8 mg of the ester in 2.5' ml of dimethylsulfoxide were then added dropwise over the course of 1 hour. The reaction solution was immediately treated with methanol/ester and the insulin derivative which precipitated was washed with methanol/ether 1:9), briefly dried in vacuo, dissolved in'a mixture of l ml of glacial acetic and 9 ml of water, and applied to a column (3 X 200 cm) of Sephadex G-50 fine in 10% strength acetic acid and chromatographed. The eluates (compare FIG. 1) where dialyzed and lyophilized.

Weighings:

Fraction 1 29 mg Fraction 2 14 mg Fraction 3 55 mg (45.8% of theory) EXAMPLE 5 N N -adi oylinsuIin (sheep) A solution of 23.3 mg (60 amol) of adipic acid bis-pnitrophenyl ester in 4 ml of dimethylsultoxide was added dropwise over the course of 4 hours at room temperature, with stirring to a solution of 320 mg of crystalline sheep insulin (50 umol) in ml of dimethylsulfoxide and ulof triethylamine. The reaction mixture was allowed to stand for a further 48- hours at room temperature and was then worked up as indicated in Example 1. The crude product was chromatog-raphed on Sephadex (column: 3 X 200 cm) as in Ex ample 1. 1

Weighings:

Fraction 1 .88 mg Fraction 2 74 mg Fraction 3 94 mg (29.4; of theory) According to quantitative paper electrophoresis. fraction 3 contained 59% ofN N adipolyinsulin.

EXAMPLE 6 N N "--pimeloylinsulin (beef) 640mg of crystalline beef insulin were reacted with 48.2 mg (120 untol) of pimelic acid bis-p-nitrophenyl cster'under the conditions described in Example 1. After 44 hours, the reaction mixture was worked up and the crude product was ehromatographed.

Weighings:

Fraction 1 Fraction 2 Fraction 3 194 mg 1 1 1 mg 288 mg (4574 of theory) 280 mg of the fraction 3 were ehromatographed on SE-Sephadex as described in Example 1. The product isolated from the main fraction (compare FIG. 3) was desalinated on Sephadex G-25.'

Yield: 132 mg (20.6% of theory) e278 5,970'(in 0.05 M ammonium bicarbonate solution, pH 8.2)

Electrophoretic purity: 95%

Free amino groups: phenylalanine Crystal form: felted needles EXAMPLE 7 N N "-suberoylinsulin' (beef) 640 mg of crystalline beef insulin were reacted with 49.9 mg umol of suberic acid bis-'p-nitrophenyl ester and worked up after 48 hours as described in Example 1. After gel filtration, the following fractions were obtained:

Fraction 1 137 mg Fraction 2 122 mg Fraction 3 331 mg (5l.7'/1-oftheory) EXAMPLE 8 'N -acelaoylinsulin (beef) 640 mg of crystalline beef insulin were reacted with 516mg (120 amol) of azclaic acid bis-p-nitrophenyl ester, as described in Example 1. After the gel filtration (see FIG. .2) the following were obtained:

Fraction 1 166 mg Fraction 2 121 mg Fraction 3 v 311 mg (489; ol'theory) EXAMPLE 9 N N -sebacoy1insulin (beef) 640 mg of crystalline beef insulin were reacted with 53.3 mg (120 ,umol) of sebacic acid bis-pnitrophenyl ester as described in Example 1 except that working up was caried out by isolating the reaction product. after dialysis, by freeze-drying. The crude product was dissolved in 4 ml of glacial acetic acid and 16 ml of water and chromatographed as in Example 1.

Weighings:

Fraction 1 175 mg Fraction 2 I43 mg Fraction 3 278 mg (43% of theory) 270 mg of fraction 3 were chromatographed on SE Sephadex as described in Example 1 and the main product (compare FIG. 3) was desalinated on Sephadex G-25.

Yield: 121.2 mg (19% of theory) Paper electrophoresis at pH 2: single substance Free amino groups: phenylalanine Crystal form: small needles EXAMPLE N N -sebacoylinsulin (pig) 640 mg of crystalline pig insulin were reacted with 53.3 mg (120 amol) of sebacic acid bis-p-nitrophenyl ester as described in Example 1. After standing for 20 hours at room temperature. the mixture was worked up as therein described.

Weighings after gel filtration:

Fraction 1 190 mg Fraction 2 139 mg Fraction 3 280 mg (43.7% of theory) Fraction 3 contained 70% of sebacoylinsulin according to quantitative electrophoretic analysis.

EXAMPLE 11 N. N 4 bis-N.N-tert.-butyloxycarbonyl )-cystinylinsulin (beef) a. A solution of 95.8 mg (120 umol) of N,N'-bistert.'butyloxycarbonyl-cystine-2,4,5-trichlorophenyl ester in 5 ml of dimethylsulfoxide was added dropwise over the course of 5 hours, with stirring to a solution of 640mg of crystalline beef insulin in 75 ml of dimethyl sulfoxide and 150 ,ul of triethylamine at room temperature. with stirring. After standing for 22 hours the reaction mixture was worked up as described in Example 1. The crude product was chromatographed on Sephadex G-50 in 10% strength acetic acid.

Weighings:

Fraction a-l l9] mg Fraction 11-2 132 mg Fraction a-3 261 mg (40.8% ol theory) b. The reaction was carried out under the same con ditions as in Example 11 (a) but using ,ul of N- methylmorpholine as the base.

Weighings:

Fraction h-l 206 mg Fraction h-Z 159 mg Fraction b-3 272 mg (42.5% of theory) 250 mg of fraction a-3 were separated on SE- Sephadex as described in Example 1. The main fraction was desalinated by chromatography on Sephadex G-25.

Yield: 103.6 mg (16.5% of theory) 6278 5,400 (in 0.05 M ammonium bicarbonate so lution, pH 8.2)

Paper electrophoresis (at pH 2): single substance Free amino groups: phenylalanine In the same manner. 91.8 mg 14.3% of theory) of (bis-butoxycarbonyl)-cystinyl-insulin were obtained from 260 mg of fraction b'3.

Paper electrophoresis (at pH 2): single substance Free amino groups: phenylalanine EXAMPLE 12 N""', N "-cystinyl-insulin (beef) 40 mg of N N -(bis-butyloxycarbonyl)cystinyl-insulin were dried for 15 hours in vacuo over phosphorus pentoxide and potassium hydroxide in a centrifuge tube. Trifluoroacetic acid 0.3 ml) was then added and the solution was allowed to stand for 1 hour at room temperature. The protein was subsequently precipitated by means of 10 ml of absolute ether and the residue collected by centrifugation and repeatedly washed with ether.

Yield after drying in vacuo over phosphorus pentoxide and potassium hydroxide: 42 mg (approximately 96% of theory);

6278 5,560 (in 0.05 M ammonium bicarbonate solution) Eleetrophoretic purity (a'tpH 2): single substance lns L0 EXAMPLE 13 N N *-(N-benzyloxycarbonyl)-glutamylinsulin (beef) A solution of 62.7 mg umol) of N-benzyloxycarbonyl-glutamic acid a, y-bis-pnitrophenyl ester was added dropwise over the course of 5 hours at room temperature, with stirring to a solution of 640 mg of crystalline beef insulin in 75 ml of dimethylsulfoxide and l 10 {L1 of N-methylmorpholine. After standing for 22 hours the mixture was worked up as described in Example 1 and subsequently chromatographed.

Weighings:

Fraction I I06 mg .Fraetion 2 13) mg Fraction 3 257 mg (40.2; oftheory) 250 mg of fraction 3 were chromatographed on SE Sephadex as described in Example 1. After desalination by chromatography on Sephadex G-25. 105 mg 16.4% of theory) of product were obtained.

Electrophoretic purity'(pH 2): 9571 Free amino groups: phenylalanine.

EXAMPLE l4 N N "-undecanedioylinsulin (beef) 640 mg of crystalline'beef insulin were reacted with 55.0 mg (120 ,u. mol) of undecane dicarboxylic acid bis-p-nitrophenyl ester. as described in Example 1 except that the reaction products were worked up 18 hours after the addition of the ester. After gel filtration (see FIG. 2) the following fractions .were obtained:

Fraction 1 I 144 mg Fraction 2 117 mg Fraction 3 31 1 mg (48.6% of theory) 300 mg of fraction 3 were chromatographed on; a 2.7 X 40 cm column of SP-Sephadex as describedin Example I, utilizing. however, 700 ml of the 2 buffer solutions. The working up of the main fraction (compare FIG. "3) after desalination yielded 139 mg of undecanedioylinsulin (22.571 of theory).

6278 5.390 (in 0.05 M ammonium bicarbonate solut ion pl-l 8.2)

Paper electrophoresis (at pH 2): single substance Free amino groups: phenylalanine Crystal form: spherical particles EXAMPLE 15 N N "-ddecanedioylinsulin (beef) Fraction 1 140 mg Fraction 2 157 mg 276 mg (43.1% of theory) Fraction 3 260 mg of fraction 3 were chromatographed on a column (2.7 X 40 cm) filled with 'SP-Sephadex as described in Example I, with, however, the volumes of the two buffer solutions being 700 ml each. Working up H N'Phe. .Cysr. .Cys .Lys NH of the main fraction (compare FIG. 3) after desalination yielded 108 mg dodecane dioylinsulin (17.9% of theory).

6278 5,870 (in 0.05 M ammonium bicarbonate solution) Paper electrophoresis (at pH 2): single substance Free amino groups: phenylalanine Crystal form: spherical particles EXAMPLE 16 N N -tridecanedioylinsulin (beef) 640 mg of crystalline beef insulin were reacted with 58.4 mg I20 amol) tridecane diacid bis'p-nitrophenyl ester. as described in Example I with work-up 18 hours after the addition of the ester. Upon gel filtration (see FIG. 2) the following were obtained:

Fraction I 143 mg Fraction-2 g u 154 mg Fraction 3 297 mg (46.471 of theory) 290 mg of fraction 3 were chromatographed on a column (2.7 X 40 cm) filled with SP-Sephadex as described in Example 1 with the volumes of the two buffer solutions being 700 ml each. The working up of the main fraction (compare FIG. 3) after desalination yielded 109 mg tridecanedioylinsulin (17.4% of theory).

5278 5,880 (in 0.05 M ammonium bicarbonate solution, pH 8.2) l

Paper electrophoresis (at pH 2 single substance Free amino groups: phenylalanine Crystal form: spherical particles What is claimed is:

l. A bifunctionally crosslinked insulin derivative in which the amino group of the A-l glycine is linked to the e-amino group of the B 29lysine by a bridge of the formula:

in which X is a carbon-carbon bond. alkylene of I to 15 carbon atoms or alkylene of l to 15 carbon atoms in which two of said carbon atoms are replaced by sulfur or wherein said alkylene is substituted at one or two of the remaining carbon atoms by an amino or amido group.

2. A monomeric A-I B-29 crosslinked insulin derivative of the formula Gly i rim WS S 'S I (IF-=0 I I (iil Cys Cys'. .Cys-- Asn x s s. l I l 4 S I (3-29) I I lt iinami-p.

wherein X is a carbon-carbon bond. an alkylene chain containing l to carbon atoms or alkylene of l to 15 carbon atoms in which two of said carbon atoms are replaced by sulfur or wherein said alkylene is substituted at one or two of the remaining carbon atoms by an amino group.

3. A compound according to claim 2 wherein X is a carbon-carbon bond or alkylene of l to l5 carbon atoms.

4. A compound according to claim 3 in which the insulin is beef insulin.

5. A compound according to claim 3 in which alkylene is of the formula (CH in which n is an integer from I to l5.

6. The compound according to claim 3 which is N. N' -oxalyl insulin.

7. The compound according to claim 3 which is N". N"--"-succinyl insulin.

8. The compound according to claim 3 which is N. N"' -glutaryl insulin.

9. The compound according to claim 3 which is N""'. N"' -adipoyl insulin.

10. The compound according to claim 3 which N N"' pimeloyl insulin.

11. The compound according N N"' "-suberoyl insulin.

12. The compound according N N"' -azelaoyl insulin.

13. The compound according N N"' -sebacoyl insulin.

14. The compound according N N" "-undecanedioyl insulin.

15. The compound according N N' -dodecanedioyl insulin.

16. The compound according N N -tridecanedioyl insulin.

17. The derivative according to claim 1 which is N, N' -(N,N-bis-tert.-butyloxycarbonyl)-L-cystinyl insulin.

18. The derivative according to claim 1 which is N N -L-cystinyl insulin I 19. The derivative according to claim 1 which is N, N N-benzyloxycarbonyl-L-glutamyl insulin.

claim 3 which is claim 3 which claim 3 which claim 3 which to claim 3 which is to claim 3 which is 20. A process for the production of a compound according to claim 1 in which insulin is reacted with an activated derivative of a dicarboxylic acid having the general formula:

in which X is a carbon-carbon double bond. alkylene of l to 15 carbon atoms or alkylene of l to 15 carbon atoms in which two of said carbon atoms are replaced by sulfur or wherein said alkylene is substituted at one or two of the remaining carbon atoms by an amino group. and Y is a carboxylic acid activating group, in a nonaqueous or aqueous polar organic solvent, in the presence of a proton acceptor and at a temperature of from about ()40 C.

21. A process according to claim 20 in which the reaction is carried out at 18 to 25C.

22. A process according to claim 20 in which L2 to L3 mole of the activated derivative are present per mole of insulin.

23. A process according to claim 20 in which Y is an unsubstituted or substituted phenoxy group.

24. A process according to claim 20 wherein impurities are removed from the derivative by sequentially differentiating the materials present firstly by their molecular weight and secondly by their charge.

25. A process according to claim 24 in which the derivative is separated from impurities by gel chromatography under conditions where insulin and insulin derivatives do not aggregate.

26. A process according to claim 23 in which the desired compound is separated from impurities by ion exchange chromatography or by electrophoresis in an acid medium.

27. A process according to claim 20 in which the derivative is produced in crystalline form by precipitation from a solution containing a zinc salt. 

1. A BIFUNCTIONALLY CROSSLINKED INSULIN DERIVATIVE IN WHICH THE AMINO GROUP OF THE A- L GLYCINE IS LINKED TO THE $AMINO GROUP OF THE B-29 LYSINE BY A BRIDGE OF THE FORMULA:
 2. A monomeric A-1, B-29 crosslinked insulin derivative of the formula
 3. A compound according to claim 2 wherein X is a carbon-carbon bond or alkylene of 1 to 15 carbon atoms.
 4. A compound according to claim 3 in which the insulin is beef insulin.
 5. A compound according to claim 3 in which alkylene is of the formula -(CH2)n-, in which n is an integer from 1 to
 15. 6. The compound according to claim 3 which is NA-1, NB-29-oxalyl insulin.
 7. The compound according to claim 3 which is NA-1, NB-29-succinyl insulin.
 8. The compound according to claim 3 which is NA-1, NB-29-glutaryl insulin.
 9. The compound according to claim 3 which is NA-1, NB-29-adipoyl insulin.
 10. The compound according to claim 3 which is NA-1, NB-29-pimeloyl insulin.
 11. The compound according to claim 3 which is NA-1, NB-29-suberoyl insulin.
 12. The compound according to claim 3 which is NA-1, NB-29-azelaoyl insulin.
 13. The compound according to claim 3 which is NA-1, NB-29-sebacoyl insulin.
 14. The compound according to claim 3 which is NA-1, NB-29-undecanedioyl insulin.
 15. The compound according to claim 3 which is NA-1, NB-29-dodecanedioyl insulin.
 16. The compound according to claim 3 which is NA-1, NB-29-tridecanedioyl insulin.
 17. The derivative according to claim 1 which is NA-1, NB-29-(N, N''-bis-tert.-butyloxycarbonyl)-L-cystinyl insulin.
 18. The derivative according to claim 1 which is N A-1, NB-29-L-cystinyl insulin.
 19. The derivative according to claim 1 which is NA-1, NB-29-N-benzyloxycarbonyl-L-glutamyl insulin.
 20. A process for the production of a compound according to claim 1 in which insulin is reacted with an activated derivative of a dicarboxylic acid having the general formula:
 21. A process according to claim 20 in which the reaction is carried out at 18* to 25*C.
 22. A process according to claim 20 in which 1.2 to 1.3 mole of the activated derivative are present per mole of insulin.
 23. A process according to claim 20 in which Y is an unsubstituted or substituted phenoxy group.
 24. A process according to claim 20 wherein impurities are removed from the derivative by sequentially differentiating the materials present firstly by their molecular weight and secondly by their charge.
 25. A process according to claim 24 in which the derivative is separated from impurities by gel chromatography under conditions where insulin and insulin derivatives do not aggregate.
 26. A process according to claim 23 in which the desired compound is separated from impurities by ion exchange chromatography or by electrophoresis in an acid medium.
 27. A process according to claim 20 in which the derivative is produced in crystalline form by precipitation from a solution containing a zinc salt. 